PROJECT SUMMARY There are fundamental gaps in our understanding of the genetics and neurobiology of DDX3X, a high-risk gene for X-linked intellectual disability (ID) accounting for up to 2% of unexplained diagnoses in females. DDX3X mutations display a puzzling sexual dichotomy: most mutations are de novo and found only in females; the few mutations in males are inherited from healthy mothers. DDX3X regulates mRNA translation, but the mechanisms of action in neurons, the target genes, and the impact of clinical mutations have not been studied. Also, the influence of sex remains unknown. There is a critical need to fill these gaps because, until we do so, implementing strategies that use DDX3X as a therapeutic target remains out of reach. The long-term goal is to map the synaptic mechanisms underlying ID and identify new therapeutic targets. The overall objective is to capture the sex-specific synaptic changes resulting from mutations in DDX3X. The central hypothesis is that DDX3X regulates synaptic translation and synaptogenesis in a sex-specific manner: mutations impact these processes differently in males and females, leading to sex biases in prevalence and severity of the condition. The rationale is that, once we know how DDX3X regulates synaptic translation and how sex influences it, mechanism-based precision therapeutics can be developed. The central hypothesis will be tested by pursuing two Specific Aims: 1) Analyze the role of DDX3X in synaptic translation and synaptogenesis; and, 2) Determine the effects of sex-specific DDX3X mutations. Under Aim 1, biochemical and structural methods will be applied to study DDX3X-mediated translation in mouse synapses. Viral-based Translating Ribosome Affinity Purification (vTRAP-seq) will be used to map the mRNAs regulated by DDX3X in male and female mouse synapses. Dendritic spines analyses in male and female single-embryo neurons from a novel DDX3X mouse model will be used to assess the role in synaptogenesis. Under Aim 2, two male- and three female-pathogenic mutations will be structurally and biochemically modeled. The mutations will be introduced in cultured neurons and the spine phenotype compared across mutations and sexes to understand why female-pathogenic mutations are more severe. All these methods are part of the applicant's expertise. This proposal is innovative because it addresses the biology of a novel ID high-risk gene in light of a central and yet overlooked aspect: sex. It is innovative because it models patient-specific mutations in neurons. It is also innovative because it integrates genetics, structural biology, biochemistry, molecular biology and neuroscience, and uses a novel mouse model and novel DDX3X small-molecule inhibitors. This application is significant because it will critically advance our understanding of DDX3X syndrome and ID more broadly. The results will expose fundamental aspects of synaptogenesis, thus advancing the knowledge of brain development in health and disease. The results are expected to have a positive impact because they will pinpoint novel molecular targets for precision medicine, while informing clinical genetics care, with both short-term and long-term benefits for families.